The circadian clock of the bacterium B. subtilis evokes properties of complex, multicellular circadian systems

Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff’s rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

The graph shows the cell number (quantified in CFUs per ml) every 4 hours during the last day of entrainment and the first day in free-run, and every 12 h afterwards until h156. Data represent averages of 3 technical replicates with SEM. B) GFP fluorescence shown for the TB269 strain (PS216 with Phy-gfp) constitutively expressing GFP (n=24). Shown are mean traces with SEM. A, B) Cultures were exposed to 3 days of entrainment with bLD cycles and subsequent release in constant darkness. Blue and grey areas indicate, respectively, the blue light and the dark phases of the zeitgeber cycles. Bacteria were grown at a constant temperature of 27 °C.    Bruce (21) showed that mice (Peromyscus) have a single bout of activity per 24h when exposed to LD cycles that are short but a harmonic of 24h (e.g., 8h).  (69)). Mice were exposed to an LD cycle length (T) of 20h or 28h (70). The free running period (FRP) of locomotor activity following release to constant conditions was significantly shorter after T20 cycles than after T28. The after-effect was transferred to pups that were born in DD from mothers previously subjected to T20h or T28h. Furthermore, after-effects were observed in vitro in the expression of the Per1-luc promotor reporter in isolated SCN tissue. Mice were exposed to T21h, T26h or T28h LD cycles (71). As in (70), aftereffects in FRP were observed for both locomotor activity and in vitro, here for PER2::LUC bioluminescence.
Aftereffects in vitro were observed for the SCN but not in peripheral tissues (spleen, esophagus, lung and thymus).
FRP of locomotor activity lengthened with increasing levels of light (72). This was observed also in retinally degenerate mice, indicating that rod and cone photoreception is not necessary for Aschoff´s rule.
The FRP of CCA1::LUC shortens with increasing light intensity under constant red or blue light ((78) , Fig. 2b). The authors found that blue light triggers the turnover of the blue light photoreceptor CRY2, in a manner that is proportional to light intensity.
The FRP becomes shorter in samples released from incubation in longer LD Tcycles (79). Clock mutant strains showed the opposite effect (longer FRP corresponding to longer Tcycles) (Fig. 3 in (79)).
L. polyedra free runs in constant blue light (82). A 4h pulse of red light lengthens the FRP of the bioluminescence glow rhythm (Fig. 6 in (82)).
The FRP of the bioluminescent glow rhythm of L. polyedra shortens under constant blue or white light, as the light intensity increases (83). Constant red or yellow light has the opposite effect ( Fig. 2 in (83)).

Bacteria; cyanobacteria
Nitrogen fixation occurs with a 24h rhythm in Cyanothece sp. exposed to 6h:6h LD cycles ( Fig.  1A in (84)). Microarray analysis revealed some genes in 6h:6h LD cycles exhibited a driven To our knowledge, no studies have been performed to explore aftereffects in cyanobacteria to date.

Bacteria; Bacillus subtilis
The expression of a reporter gene PytvA-lux displays frequency demultiplication in T12h symmetrical cycles of blue light/darkness (This study, Fig. 3).
FRP of the PytvA-lux reporter changes systematically when cultures are released to DD from bLD zeitgeber cycles of different amplitudes.
The FRP becomes progressively shorter as cultures are entrained to bLD cycles using higher fluence rate (This study, Fig. 4).